AIMS: Intramuscular fat contributes to peripheral insulin resistance and type 2 diabetes mellitus (T2DM). Intra- and extramyocellular lipids (IMCLs&EMCLs) may be quantified by magnetic resonance imaging (MRI) and serve as imaging biomarkers in impaired glucose metabolism. MATERIALS & METHODS: Subjects from a population-based cohort were classified with T2DM, prediabetes or normoglycemic controls. Total myosteatosis, IMCLs and EMCLs were quantified by Multi-echo Dixon MRI as proton-density fat-fraction (PDFF in %) in abdominal skeletal muscle. RESULTS: Among 337 included subjects (median age 56.0years (IQR:49.0-64.0years), 56.4% males, median BMI:27.2kg/m2 ) 129(38.3%) were classified with an impaired glucose metabolism (T2DM: 49(14.5%); prediabetes: 80(23.7%)). IMCLs were significantly higher than EMCLs in subjects without obesity (5.7%(IQR: 4.8-7.0%) vs. 4.1%(IQR: 2.7-5.8%), p<0.001), whereas the amount of IMCLs and EMCLs was shown to be equal and significantly higher in subjects with obesity (both 6.7%, p<0.001). Subjects with prediabetes and T2DM had significantly higher amounts of IMCLs and EMCLs as compared to normoglycemic controls (p<0.001). In univariable analysis, prediabetes and T2DM were significantly associated with both, IMCLs (prediabetes: β:0.76, 95%-CI:0.28-1.24, p=0.002; T2DM: β:1.56, 95%-CI:0.66-2.47, p<0.001) and EMCLs (prediabetes: β:1.54, 95%-CI:0.56-2.51, p=0.002; T2DM: β:2.15, 95%-CI:1.33-2.96, p<0.001). After adjustment for age and gender, the association of IMCLs with prediabetes attenuated (p=0.06), whereas for T2DM, both IMCLs and EMCLs remained significantly and positively associated (p<0.02). CONCLUSION: There are significant differences in the amount and distribution ratio of IMCLs and EMCLs between subjects with T2DM, prediabetes and normoglycemic controls. These patterns of intramuscular fat distribution by MRI might therefore serve as imaging biomarkers in both normal and impaired glucose metabolism.